Abstract

For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.

(A) Molecular representation of the hydrated streptavidin tetramer. (B) Projection map (32×32 pixels with a pixel size of 0.182 nm); (C) Projection map was Gaussian-filtered with a half power at 1/1.3 nm−1; (D) Data-derived projection map by merging reflections computed from two images taken at 0.5 and 4.5 μm defocus (shown in ) to a resolution of 1/1.3 nm−1. (E) Projection map from electron crystallography (), also shown in (F), Gaussian-filtered with a half power at 1/1.3 nm−1. The images are 5.82 nm square.

Tethering of liposomes onto 2D crystals. Cryo-EM images of liposomes with no supporting structures (A) and with the 2D crystal nano-support (B). (C) Scale drawing of the tethering system. The 2D crystal layer (5.0 nm) is bound to a lipid monolayer (2.5 nm) which spans a hole in the perforated carbon film (15–30 nm in thickness). A liposome with a small diameter (45 nm) is diagrammed here. Liposomes prefer to stay near the edge of the holes in regular cryo-EM samples. When the 2D streptavidin crystal was used, a homogeneous distribution of liposomes was obtained.

Liposome tethering kinetics. EM images of negatively stained 2D streptavidin crystal (spanning holes in the perforated carbon film) with tethered liposomes (POPC liposomes doped with biotin-DPPE) at low (A), intermediate (B) and high (C–D) magnification. The incubation time in (C) and (D) are 0.5 and 1.0 min respectively. (E) EM images of negatively stained liposomes on a glow-discharged continuous carbon film with an incubation time of 4.0 min. (F) The dependence of the average liposome density on the incubation time. The lines represent the liposome density on 2D crystal (solid line) and carbon film (dashed line), respectively.

Protein concentration and crystal growth time effects on the crystal size. EM images show the negatively stained crystal grown from (A) 0.2 mg/ml (B) 0.1 mg/ml (C) 0.05 mg/ml of streptavidin solutions for 2 hours at room temperature. The black curves outline the domain boundaries, the arrow shows the crystal orientation in each region, and each inset is a 3× closeup of the adjacent region. (D) The dependence of average single crystal size on the protein concentration and growth time. The crystal growth time was varied from 2 hr (dashed line) to 6 hr (solid line) and overnight (data not shown, but the crystal covered entire 2 μm-diameter holes for 0.2 mg/ml of protein concentration). The error bar is the standard deviation of crystal sizes.

Effect of the crystal lattice removal process on the images of DPhPC liposomes tethered on the 2D crystal. (A) Cryo-EM image of liposomes tethered to a 2D crystal. (B–C) Cryo-EM image of an individual liposome before and after the removal of crystal information. The images were Gaussian-filtered (half power at 2.0 nm) for display. (D–E) The center quarter of the power spectrum of the image before and after masking the spots. (F) Circularly averaged experimental liposome image contrast w (dots) compared with model (line). The image was taken on film, without energy filtering, using the Tecnai F20 electron microscope at 200 keV. The defocus was 2.20 μm and the B factor was estimated to be 1.86 nm2.

Crystal analysis. (A–B) Overview of images (529 nm square) at 0.52 and 4.54 μm defocus. (C–D) Corresponding close-up of 20×20 unit cells; (E–F) Displacements of centers of arrays of 2×2 unit cells from positions predicted based on a perfect lattice. Vectors are magnified by a factor of 50. (G–H) IQ plots. The solid circles show resolution of 2.0 and 1.0 nm, and the circles in thin line show the CTF zero positions. The size of the symbols is proportional to the strength of the spot, and the numbers are IQ values. The definition of IQ follows Henderson et al (). As a guideline, an IQ number of 7 (marked by the smallest symbols) is equivalent to a signal-to-noise ratio (SNR) of 1, and an IQ number of 2 is equivalent to a SNR of 3.5. The SNR was calculated as the ratio of the spot amplitude to the rms background value.

Determination of m factors. (A–B) Comparison of the computed reflections from cryo-EM images taken at 0.52 and 4.54 μm defocus and from the model. The real parts of the observed Fourier amplitudes were flipped according to the polarity of the model structure factors, resulting in polarities that reflect the alternating polarities of the CTF. Model structure factors were multiplied by the CTF (with B set to zero). The labels are reflection indices, and the numbers in parenthesis are the spatial frequencies in units of nm−1 corresponding to the reflections. The oval in (B) marks reflection spots having inverted phases compared with the model. (C–D) Contrast-transfer function c corresponding to the images above. The red crosses mark the spatial frequencies of the major reflections and demonstrate the polarity reversals. (E–F) Determination of m0and B. The solid line is the fitted Gaussian function, with B = 1.42 and 1.54 nm2 respectively. The labels are spatial frequencies in nm−1, and in parentheses are the numbers of computed reflections averaged in each group.

Analysis of m factors and CTF parameters. (A) m factors from 9 cryo-EM images recorded on a CCD camera using a Tecnai F30 with the defocus from 0.5 to 4.7 μm. The B factor determined from the least-squares fit of a Gaussian function (solid line) is 1.55 nm2. (B) Values of m in four frequency bands (indicated in panel A) as a function of ice thickness, for which the logarithm of beam attenuation serves as a surrogate. Ice thickness does not seem to affect m in these images obtained with an energy filter. (C) Comparison of defocus values determined from crystals and from carbon film. (D) Comparison of B factors determined from crystal and carbon film. The data for (C) and (D) was collected using the Tecnai F20 microscope and recorded on film.